The capability to (1) systematically collect, process, and archive nucleic acids from "extremely low-biomass" spacecraft-related environments, and (2) effectively assess the diversity of microorganisms present on spacecraft and associated cleanroom surfaces was developed, and validated. This capability enabled generation of the most comprehensive (bacterial, archaeal, and fungal) assessment of spacecraft-associated biodiversity to date. The capability to provide a passenger list of the microorganisms associated with flight hardware developed and validated in this study bridges a significant gap in technology and dramatically increases NASA's ability to explore and verify the scientific findings of both in-situ life detection and sample-return missions.

Although there is a growing understanding of the biodiversity associated with low biomass surfaces, it remains challenging to provide a comprehensive inventory of microbes present. In this study three molecular approaches were attempted: conventional cloning techniques, PhyloChip DNA microarrays, and 454 tag-encoded pyrosequencing, together with a methodology to systematically collect, process, and archive nucleic acids, to assess the phylogenetic breadth of microorganisms present on spacecraft and associated surfaces. The analysis methods yielded very different results; traditional approaches provided the least comprehensive assessment of microbial diversity, while PhlyoChip and pyrosequencing detected more diverse microbial populations. The findings of this pioneering study provided new and important insights into the benefits and limitations of modern molecular approaches for assessing the microbial diversity associated with samples extremely low in total biomass. These are of particular relevance to current and future NASA endeavors, as well as homeland security, medical, pharmaceutical, and semiconductor applications.

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